Chapter 20 - Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplification for Improved Protein Analyses

Abstract

While mass spectrometry has emerged as a powerful research tool for mapping the protein composition of biological samples, assays based on reagents with affinity for specific proteins remain the most promising avenue for protein-based diagnostics. In proximity ligation assays (PLA), single-stranded DNA oligonucleotides are conjugated to antibodies, and the conjugates are referred to as proximity probes. This chapter provides an overview of current methods for protein analysis, as a background to the description of PLA, which is a general molecular strategy for protein analysis. The technique examines interactions between proteins and specific DNA sequences, illustrating the versatility of this new detection mechanism. This approach is complicated by the difficulty of generating suitable affinity reagents. Multiple proteins can potentially be investigated in parallel without rapidly growing problems with cross-reactivity by ensuring that only cognate pairs of proximity probes give rise to detection signals. Proximity ligation lends itself to assays where protein levels are measured and where their distribution is visualized in tissues, illustrating functional effects of signal transduction, developmental processes, as well as effects of disease and of therapeutic intervention. Future developments of this technique are discussed. At this point it is not clear what level of multiplexing of protein measurement can be achieved by proximity ligation but the assay is compatible with a wide range of readout formats, including separate real-time amplification reactions or novel microarray detection mechanisms.